This invention relates to the construction and characterization of mouse monoclonal antibodies against western equine encephalitis virus (WEE) expressed from hybridoma cell lines.
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Western equine encephalitis virus (WEE) is an enveloped positive-sense, single-stranded RNA virus belonging to the alphavirus genus. The 12 kb genome of WEE encodes for nonstructural (5xe2x80x2 end) and structural (3xe2x80x2 end) proteins. The structural proteins are translated from a subgenomic mRNA (26S mRNA) as a polyprotein that is processed by viral and cellular proteases into E1 (53 kDa), E2 (47 kDa), nucleocapsid [NC] (30 kDa), E3 (10 kDa), and 6K (6 kDa) proteins. The E1 and E2 proteins are glycoproteins present in the lipid envelope. The E3 protein is also a glycoprotein that is most often not a component of the virion, but is required for infectivity in wild-type virus. The NC protein encloses the RNA genome in an icosahedral structure. The 6K protein is virion associated and promotes efficient virus assembly (reviewed in Strauss and Strauss, 1994; Strauss et al., 1995; Johnston and Peters, 1996; Schlesinger and Schlesinger, 1996).
WEE is localized to the Western hemisphere and poses a serious hazard to human health. Virus transmission is by infected mosquitoes, causing disease in humans and horses. Symptoms of WEE infection in humans include encephalitis, convulsions, paralysis, malaise, fever, headaches, nausea, and vomiting. The case fatality rate in humans is 2% to 7%. Currently, there are no known antiviral drugs effective against WEE. Although inactivated WEE vaccine exist for use in limited populations such as laboratory personnel who are at high risk of exposure to the virus, the immunogenicity of the inactivated WEE vaccine is often poor and the immunity is short-lived. Better protection against WEE is required (Johnston and Peters, 1996).
Alphavirus antigenic properties and antibody neutralization have been studied with anti-alphavirus antibodies from mouse immunoglobulins. Murine antibodies capable of neutralizing virus have been generated against E1 and E2 (Mathews and Roehrig, 1982; Boere et al., 1983; Yamamoto et al., 1985; Yamamoto, 1986). Mice were protected from challenge with WEE and Venezuelan equine encephalitis virus (VEE) when injected with antibodies against E1 and E2 in passive immunization studies (Mathews and Roehrig, 1982; Hunt and Roehrig, 1985; Yamamoto, 1986). Anti-E2 monoclonal antibodies were able to protect mice from lethal injections of Semliki Forest virus (SFV) (Boere et al., 1983). Furthermore, neutralizing and non-neutralizing antibodies to E1 and E2 administered to mice, before or after infection with virus, were protected from Sindbis virus (SIN) (Griffin et al., 1997).
Animal antisera and monoclonal antibodies provide important sources of antibody. Although recombinant antibodies have the advantages of being produced quickly, economically, and in large quantities (Wright et al., 1992; Hayden et al., 1997; Verma et al., 1998), recombinant antibodies grown in bacterial systems are often improperly folded and nonglycosylated (Wright et al., 1992; Verma et al., 1998). One may favor the use of monoclonal antibodies over recombinant antibodies for a variety of reasons. Hybridoma technology is able to provide a wide range of monoclonal antibodies that bind to different antigens with high specificity and affinity (Winter and Milstein, 1991; Laurino et al., 1999). Furthermore, monoclonal antibodies can be isolated with high purity (Winter and Milstein, 1991; Laurino et al., 1999). Accordingly, production of monoclonal antibodies directed against WEE is desirable.
Up until recently, only a limited number of monoclonal antibodies against WEE existd and have not been fully characterized. For instance, monoclonal antibodies produced by Hunt and Roehrig (1985) are capable of immunoprecipitating the E1/E2 heterodimer, identifying antigenic determinants on E1, and protecting mice when challenged with WEE. Monoclonal antibodies produced by Yamamoto et al. (1985), showing specificity for E1 and E2 in enzyme-linked immunosorbent assays (ELISA), demonstrate neutralizing activity and are found effective in passive immunization studies (Yamamoto et al., 1985; Yamamoto, 1986). Recently, there have been studies directed to specific recombinant antibodies against WEE. For example, Xu et al. (1999) successfully cloned an anti-WEE scFV. In addition, use of recombinant antibodies to histologically stain the cells expressing WEE antigens was reported in Netolitzky et al. (2000). Accordingly, it is advantageous to produce and characterize a group of monoclonal antibodies for use in detecting and diagnosing WEE effectively. It is also advantageous to study the interactions between monoclonal antibodies with other alphaviruses, such as VEE and SIN.
The present invention is directed to the construction and characterization of a group of mouse monoclonal antibodies against WEE.
An object of the present invention is to produce and identify specific monoclonal antibodies displaying various immunological activities against WEE.
Another object of the present invention is to construct and characterize monoclonal antibodies capable of cross-binding to multiple alphaviruses.
It is another object of the present invention to manufacture recombinant antibodies for hydridoma clones expressing anti-WEE monoclonal antibodies.
It is yet a further object of the present invention to use the identified monoclonal antibodies for immunodetection and immunotherpy.
According to one aspect of the present invention, it provides monoclonal antibodies against WEE expressed from hybridomas.
According to another aspect of the present invention, it provides for the construction of recombinant monoclonal antibodies from hybridoma clones against WEE, consisting of the steps of immunizing mice with antigens prepared from WEE infected cells; fusing and cloning hydridoma cells lines from the immunized mice; and genetic engineering recombinant antibodies from said cultured hybridoma cells.